Nucleotide based endonucleases

ABSTRACT

This invention provides catalytic molecules capable of cleaving target nucleotide sequences. More specifically, the invention provides an endonuclease having nucleotide sequences which are of sufficient length to allow hybridisation to a target nucleotide sequence desired to be cleaved. The endonuclease contains a catalytic region comprising ribonucleotides and/or deoxyribonucleotides, or derivatives thereof which act to cleave a phosphodiester bond of the substrate nucleotide sequence. The catalytic region comprises nucleotides or derivatives thereof which are linked by linking groups which may comprise ribonucleotides, deoxyribonucleotides or combinations thereof. 
     The endonucleases of the invention are useful in the cleavage of target RNAs associated with disease in humans and animals and in the inactivation of RNA transcripts in eukaryotic and prokaryotic cells, as well as the cleavage of RNA transcripts in-vitro.

This application is a continuation of U.S. Ser. No. 08/218,656, filedMar. 28, 1994, which is a continuation of U.S. Ser. No. 07/717,602,filed Jun. 19, 1991, now U.S. Pat. No. 5,298,612, issued Mar. 29, 1994.

This invention generally relates to endonucleases which are capable ofcleaving nucleic acids; vectors encoding endonucleases; host cells andorganisms either modified by and/or containing or encoding nucleic acidendonucleases; and to methods for the cleavage and/or inactivation ofnucleic acid molecules in-vivo or in-vitro.

FIELD OF THE INVENTION

There have previously been described catalytic endoribonucleasescomprised of RNA which are capable of effecting the cleavage of targetRNA. Such endoribonucleases generally fall into two categories. Thefirst, are based on the mitochondrial intervening sequence (IVS) RNA ofthe organism tetrahymena, such as described by Zaug et al. (Nature, Vol.324, 429-433, 1986). The second class of endoribonucleases are theresult of pioneering work by Haseloff and Gerlach (Nature, Vol. 334,585-591, 1988) on the self-cleaving regions of plant viral RNA.

This invention is directed to hitherto unknown endonucleases.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A-1B (SEQ ID NOS.1-7) shows base sequences, schematicrepresentations and the names of various endonucleases. Conservedribonucleotides are depicted by bold type and thick lines. Otherribonucleotides are depicted by lower case letters and wavy lines. Thenomenclature for the endonucleases is as follows: R indicates a ribozymecontaining helix II, M denotes an endonuclease not containing helix II,and MS denotes an endonuclease and substrate in the same molecule; thenumbers and nucleotide designations indicate the bases in the connector,and the RNA or DNA refers to the nucleotides in the arms that formhelices I and III with the substrate.

SUMMARY OF THE INVENTION

In accordance with a first aspect of this invention, there is providedan endonuclease of the formula (I)

    X--M--Y                                                    (I)

wherein X and Y represent nucleotide sequences comprised ofdeoxyribonucleotides, ribonucleotides, or combinations thereof orderivatives thereof; said nucleotide sequences being of sufficientlength to allow hybridization to a target nucleic acid sequence desiredto be cleaved by said endonuclease;

and wherein M represents a catalytic region of the formula (II):##STR1##

where, A, G, C and U respectively represent bases adenine, guanine,cytosine and uracil which may be in the form of deoxyribonucleotides,ribonucleotides, or combinations thereof or derivatives thereof; and N¹²is selected from any of the bases adenine, guanine, cytosine, uracil orthymine, or derivatives thereof;

and wherein;

P is one or more nucleotides, which nucleotides may bedeoxyribonucleotides, ribonucleotides, or a combination of one or moredeoxyribonucleotides and one or more ribonucleotides, or derivativesthereof, wherein if said nucleotides solely comprise ribonucleotides andthe nucleotide sequences X and Y are solely comprised ofribonucleotides, then said ribonucleotides of the group P are not basepaired to one another; or a bond or any atom or any group ofinterconnected atoms linking nucleotides A⁷ and G⁸, which does notdestroy the cleavage capability of the catalytic region and which is notsolely comprised of nucleotides.

For the sake of convenience, such endonucleases may be referred to asminizymes.

In the catalytic region M, nucleotide additions, deletions orreplacements may be made with the proviso that endonuclease activity isnot destroyed. For example, any one of nucleotides C¹ to A¹¹ may besubstituted with one or more ribo- and/or deoxyribonucleotidescontaining bases such as adenine, guanine, cytosine, methyl cytosine,uracil, thymine, xanthine, hypoxanthine, inosine, or other methylatedbases. Nucleotide bases and deoxynucleotide bases are well known in theart and are described, for example in Principles of Nucleic AcidStructure (Ed, Wolfram Sanger, Springer-Verlag, New York, 1984) which isincorporated herein in its entirety by reference. Nucleotides C¹ to N¹²may be substituted with any ribonucleotide or deoxyribonucleotide knownper se, with the proviso that endonuclease activity, particularlyendoribonuclease activity, is not lost. Endoribonuclease activity may bereadily and routinely assessed as will be described hereinafter.

It is preferred that the catalytic region be comprised ofribonucleotides. Notwithstanding this, one or more of the nucleotides C¹to A¹¹ of the catalytic region may be in the form ofdeoxyribonucleotides as long as endoribonuclease activity is not lost.

Ribonucleotide and deoxyribonucleotide derivatives or modifications arewell known in the art, and are described, for example, in Principles ofNucleic Acid Structure (Supra, particularly pages 159-200), and in theCRC Handbook of Biochemistry (Second edition, Ed, H. Sober, 1970) whichis incorporated herein by reference.

Nucleotides comprise a base, sugar and a monophosphate group.Accordingly, nucleotide derivatives or modifications may be made at thelevel of the base, sugar or monophosphate groupings.

A large number of modified bases are found in nature, and a wide rangeof modified bases have been synthetically produced (see Principles ofNucleic Acid Structure and CRC Handbook of Biochemistry, Supra). Forexample, amino groups and ring nitrogens may be alkylated, such asalkylation of ring nitrogen atoms or carbon atoms such as N₁ and N₇ ofguanine and C₅ of cytosine; substitution of keto by thioketo groups;saturation of carbon═carbon double bonds, and introduction of aC-glycosyl link in pseudouridine. Examples of thioketo derivatives are6-mercaptopurine and 6-mercaptoguanine.

Bases may be substituted with various groups, such as halogen, hydroxy,amine, alkyl, azido, nitro, phenyl and the like.

The sugar moiety of the nucleotide may be modified according to wellknown methods in the art (see Principles of Nucleic Acid Structure andCRC Handbook of Biochemistry, Supra). This invention embraces variousmodifications to the sugar moiety of nucleotides as long as suchmodifications do not abolish cleavage activity of the endonuclease.Examples of modified sugars include replacement of secondary hydroxylgroups with halogen, amino or azido groups; 2'-methylation;conformational variants such as the 02'-hydroxyl being cis-oriented tothe glycosyl C_(1') -N link to provide arabinonucleosides, andconformational isomers at carbon C_(1') to give α-nucleosides, and thelike.

The phosphate moiety of nucleosides is also subject to derivatisation ormodifications, which are well known in the art. For example, replacementof oxygen with nitrogen, sulphur or carbon derivatives to respectivelygive phosphoramidates, phosphorothioates and phosphonates. Substitutionsof oxygen with nitrogen, sulphur or carbon derivatives may be made inbridging or non bridging positions. It has been well established fromwork involving antisense oligonucleotides that phosphodiester andphosphorothioate derivatives may efficiently enter cells (particularlywhen of short length), possibly due to association with a cellularreceptor. Methylphosphonates are probably readily taken up by cells byvirtue of their electrical neutrality.

Deoxyribonucleotide or ribonucleotide derivatives as referred to in thisspecification embody one or more of the modifications referred to abovewhich do not destroy the cleavage capability of the endonuclease.

Bases and/or nucleotides 1 to 11 of the catalytic region may besubstituted with other chemical species, such as an amino-acid sidechain or linkers which may or may not incorporate other chemicalentities, e.g. acidic or basic groups. For example, G³ may besubstituted with tyrosine, and C¹ or A¹¹ similarly substituted withhistidine. In some instances it may prove possible to delete afunctionally important chemical species (e.g., nucleotide or amino-acidside chain) and provide this as part of the substrate or as a co-factorwhich transactivates the endonuclease. Such derivatives which possessendonuclease activity are within the scope of the present invention.

Endonuclease activity is readily, simply and routinely tested byincubating the endonuclease with its substrate and thereafter assessingwhether cleavage of the substrate takes place. For example, cleavage ofa target mRNA takes place after the trinucleotide sequence X'UY' whereX' and Y' represent any ribonucleotide, and which may be the same ordifferent and U represents a ribonucleotide having the base uridine.Preferred cleavage sites include GUC, GUU, GUA and UUC. By way ofexample, suitable reaction conditions may comprise a temperature fromabout 4° C. to about 60° C. (preferably about 20 to 55° C.), pH fromabout 7.0 to about 9.0 and salt (such as Mg²⁺) from about 1 to about 100mM (preferably 1 to 20 mM). Endonucleases containing a small number ofnucleotides in each of the groups X and Y of formula (I) (such as fournucleotides) in each of groups X and Y would generally be incubated atlower temperatures, such as about 20° C. to about 25° C. to aidduplexing of complementary nucleotide sequences in the endonucleasesequences X and Y and the substrate. The endonuclease would generally bein an equimolar ratio to the substrate or in excess thereof. However, asthe endonuclease may act as an enzyme, cleaving substrate withoutconsumption, the ratio of endonuclease to substrate is not ofimportance.

A target RNA containing a suitable cleavage site as mentioned above,such as GUC site may be incubated with an endonuclease which, forexample, may contain one or more modifications within the catalyticregion. The nucleotide sequences X and Y of the formula (I) are selectedso as to be complementary (that is, capable of forming base pairs) tonucleotide sequences flanking the cleavage site in the target RNA. Onincubation of the endonuclease and its substrate an enzyme/substratecomplex is formed, as a result of base pairing between complementarynucleotides in the endonuclease and the substrate. Nucleotide sequencesX and Y of the formula (I) and nucleotide sequences flanking thecleavage site in the substrate form a double stranded duplex as a resultof base pairing, which base pairing is well known in the art (Sambrook,J. et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, ColdSpring Harbor Press, 1989, which is incorporated herein by reference).The formation of a double stranded duplex between complementarynucleotides may be referred to as hybridization (Sambrook, et al.,Supra). Hybridization or duplex formation between the endonuclease andits substrate can be readily assessed, for example, by labelling one orboth components, such as with a radiolabel, and then subjecting thereaction mixture to polyacrylamide gel electrophoresis undernon-denaturing conditions (Sambrook et al., Supra). If the target iscleaved on incubation with the endonuclease it is active and is withinthe scope of this invention. Accordingly, an endonuclease containing anucleotide derivative may be simply tested for endonuclease activity ina routine manner.

As will be readily appreciated by workers in the field to which thisinvention relates, the cleavage of a target RNA may be readily assessedby various methods well known in the art (for example, see Sambrook etal., Supra). Cleavage may, for example, be assessed by running thereaction products (where the substrate is radioactively labelled) onacrylamide, agarose, or other gel systems, and then subjecting the gelto autoradiography or other analytical technique to detect cleavagefragments (Sambrook et al., Supra).

Where the group P represents a bond it may represent a chemical bond oratom or group of interconnected atoms between nucleotides A⁷ and G⁸. Thebond between nucleotides is between the base or sugar moiety, i.e.,sugar to sugar, base to sugar, or base to base. Inter base crosslinkingis described, for example, by Petric et al. (1991), Nucleic Acid. Res.,19: 585, which is incorporated herein by reference. The bond may extendfrom any position on the base or sugar ring or from any functional groupon the base or sugar ring, again with the proviso that the endonucleaseis capable of substrate cleavage.

The group P of the formula (II) may represent one or more nucleotides,which nucleotides may be deoxyribonucleotides, ribonucleotides, or acombination of one or more deoxyribonucleotides or one or moreribonucleotides, or derivatives thereof, wherein said derivatives are asdescribed herein. Where P comprises a nucleotide sequence, thenucleotide sequence may comprise a ribonucleotide sequence or acombination of one or more ribonucleotides and one or moredeoxyribonucleotides, in which none of these bases are paired, or atleast one of the bases is base paired (i.e., this including where all ofthe bases are base paired, and where not all of the bases are paired).Alternatively, the group P may comprise a deoxyribonucleotide sequencein which none of the bases are paired or at least one of the bases arepaired (again this including where all of the bases are base paired, andwhere not all of the bases are base paired). Where the nucleotidesequences X and Y are comprised solely of ribonucleotides, and the groupP is comprised solely of ribonucleotides, the ribonucleotides of thegroup P are not base paired to one another. Optionally, where nucleotidesequences X and Y are comprised solely of deoxyribonucleotides, and thegroup P is comprised solely of deoxyribonucleotides, thedeoxyribonucleotides of the group P may not be base paired.

Applicants have found that base pairing in the group P is not requiredfor cleavage of a target RNA. Accordingly, when nucleotide sequences Xand Y are comprised solely of ribonucleotides and the group P iscomprised solely of ribonucleotides, the ribonucleotides of the group Pmay be base paired for purposes other than to effect cleavage of atarget RNA. Such purposes would include to allow the binding of cellularfactors, such as RNA binding proteins or other cellular factors.Similarly, where the nucleotide sequences X and Y are comprised solelyof deoxyribonucleotides, and the group P is comprised solely ofdeoxyribonucleotides, the deoxyribonucleotides of the group P may bebase paired for purposes other than involvement in endonucleasecleavage, such as interaction with DNA binding proteins and othercellular factors, which may, for example, effect cellular distributionof the endonuclease.

Where deoxyribonucleotides, ribonucleotides, combinations thereof, andderivatives thereof, are said to be not base paired, a person ofordinary skill in the art will understand this to mean that thenucleotides are not base paired to one another according to knownnucleotide base pairing, namely Watson-Crick base pairs and Hoogsteenbase pairs, and the like (Principles of Nucleic Acid Structure, Supra).

The absence of base pairing is an advantageous feature of this inventionas endonucleases comprising a minimal number of nucleotides may beproduced according to standard methods hereinafter described. Theapplicants have surprisingly discovered that base pairs betweennucleotides in the group P are not required to permit the endonucleaseof this invention to cleave its target substrate. Accordingly, the groupP may comprise any number of non base paired nucleotides, for example,two nucleotides (such as TT), four nucleotides (such as AAAA, UUUU,TTTT, etc.) or five nucleotides (such as TTTTT). The nucleotide sequenceof the group P under these circumstances is not of importance and thenumber of nucleotides is also not of importance. Generally speaking, thegroup P may comprise from 1 to 20 non base paired nucleotides and morepreferably may contain from 2 to 6 non base paired nucleotides. The mainconsideration to take into account is that the resultant endonuclease iscapable of substrate cleavage. This can be readily measured withoutundue experimentation in standard cleavage assays on an appropriatetarget nucleotide sequences as hereinbefore described.

The group P in formula (II) may comprise ribonucleotides,deoxyribonucleotides, or at least one deoxyribonucleotide and at leastone ribonucleotide, or derivatives thereof wherein all nucleotides arebase paired, or not all of the nucleotides are base paired, aspreviously described. Complementary nucleotides in the nucleotidesequence P linking nucleotides A⁷ and G⁸ of the formula (II), may bebase paired by Watson-Crick base pairs, Hoogsteen base pairs, or otherbase pairing known in the art. Where the nucleotide sequence of thegroup P is partly base paired (that is, not all nucleotides are basepaired), there may be provided regions of base pairing and one or moresingle-stranded regions, for example, a base paired stem and a loop ofnon base paired nucleotides. For example, a stem and loop arrangement isdescribed by Haseloff and Gerlach (Supra), and has the followingsequence: ##STR2##

A sequence forming such a stem/loop structure may, in accordance withthis invention, be comprised of deoxyribonucleotides, ribonucleotides,or a combination of deoxyribonucleotides and ribonucleotides.

Notwithstanding the above, it is preferred that the nucleotides of thegroup P are not base paired. The absence of a base paired structure mayreduce steric interference of the endonuclease with its substrate orother nucleic acid sequences (thus increasing endonuclease activity),and may reduce the likelihood of non-favourable base pairinginteractions.

The group P of the formula (II) may comprise a nucleotide sequence asherein described wherein one or more ribonucleotides and/ordeoxyribonucleotides are replaced with a linker which connects adjacentnucleotides. Any chemical linker, that is any group of interconnectedatoms, may be used to link nucleotides A⁷ and G⁸, or any one ofnucleotides C¹ to A⁷ with any one of nucleotides G⁸ to A¹⁰ wherein theendonuclease is capable of substrate cleavage. Examples of such linkersare described by Petric et al., Supra). Substrate cleavage may bereadily assessed by simply incubating an endonuclease with its substrateas described hereinbefore.

One or more ribonucleotides and/or deoxyribonucleotides of the group Pmay be replaced, for example, with a linker selected from optionallysubstitited polyphosphodiester (such as poly(1-phospho-3-propanol)),optionally substituted alkyl, optionally substituted polyamide,optionally substituted glycol, and the like. Optional substituents arewell known in the art, and include alkoxy (such as methoxy, ethoxy andpropoxy), straight or branch chain lower alkyl (such as C₁ -C₅ alkyl),amine, aminoalkyl such as amino C₁ -C₅ alkyl), halogen (such as F, Cland Br) and the like. The nature of optional substituents is not ofimportance, as long as the resultant endonuclease is capable ofsubstrate cleavage.

Additionally, suitable linkers may comprise polycyclic molecules, suchas those containing phenyl or cyclohexyl rings. Such compounds wouldgenerally comprise suitable functional groups to allow coupling throughreactive groups on nucleotides.

The nucleotides of the groups X and Y may be of any length and sequencesufficient to enable hybridization formation with complementarynucleotides in the target RNA, as described herein. The nucleotides maybe in the form of deoxyribo- nucleotides, ribonucleotides,deoxyribonucleotide ribonucleotide hybrids, or derivatives thereof ashereinbefore described. These flanking sequences may be chosen tooptimize stability of the endonuclease from degradation. For example,deoxyribonucleotides are resistant to the action of ribonucleases.Modified bases, sugars or phosphate linkages of nucleotides, such asphosphoramidate, or phgosphorothioate linkages in the phosphate backboneof the nucleotide sequences, may also provide resistance to nucleaseattack. Binding affinity may also be optimised in particularcircumstances, by providing nucleotides solely in the form ofribonucleotides, deoxyribonucleotides, or combinations thereof. In somecircumstances it may be necessary to optimise the composition of thegroups X and Y, to maximize target RNA cleavage. The cleavage activityof endonucleases having flanking nucleotide sequences which hybridise totarget sequences and which are comprised wholly of deoxyribonucleotidesmay, in some circumstances, have reduced activity. In such circumstancesoptimisation may involve providing a mixture of deoxyribonucleotides andribonucleotides in the nucleotide sequences X and Y. For example,nucleotides in the endonuclease which are proximal to the cleavage sitein a target RNA may be in the form of ribonucleotides. The nucleotidesA¹¹ and N¹² of the formula (II) interact with the target sequenceadjacent to the cleavage site, with A¹¹ interacting with the U of thetarget sequence X'UY', where X' and Y' are as herein defined. Thenucleotide N¹² is selected to be complementary to the nucleotiderepresented by X'. These nucleotides, or nucleotides in a 5' directionmay, for example, be in the form of ribonucleotides. Where a targetsequence is shown to be relatively resistant to certain embodiments ofendonucleases of this invention, it may be necessary to providenucleotide sequences X and Y partly or wholly in the form ofribonucleotides. Where desired, protection from nuclease attack as willbe hereinafter described.

The respective 5' and 3' termini of the groups X and Y may be modifiedto stabilise the endonuclease from degradation. For example, blockinggroups may be added to prevent terminal nuclease attack, in particular3'-5' progressive exonuclease activity. By way of example, blockinggroups may be selected from optionally substituted alkyl, optionallysubstituted phenyl, optionally substituted alkanoyl. Optionalsubstituents may be selected from C₁ -C₅ alkyl; halogen such as F, Cl orBr; hydroxy; amino; C₁ -C₅ alkoxy and the like. Alternatively,nucleotide analogues such as phosphothioates, methylphosphonates orphosphoramidates or nucleoside derivatives (such as α-anomers of theribose moiety) which are resistant to nuclease attack may be employed asterminal blocking groups.

Alternatively, groups which alter the susceptibility of the endonucleasemolecule to other nucleases may be inserted into the 3' and/or 5' end ofthe endonuclease. For example, 9-amino-acridine attached to theendonuclease may act as a terminal blocking group to generate resistanceto nuclease attack on the endonuclease molecules and/or as anintercalating agent to aid endonucleolytic activity. It will be readilyappreciated that a variety of other chemical groups, e.g. spermine orspermidine could be used in a related manner.

Endonucleases of this invention may be covalently or non-covalentlyassociated with affinity agents such as proteins, steroids, hormones,lipids, nucleic acid sequences, intercalating molecules (such asacridine derivatives, for example 9-amino acridine) or the like tomodify binding affinity for a substrate nucleotide sequence or increaseaffinity for target cells, or localisation in cellular compartments orthe like. For example, the endonucleases of the present invention may beassociated with RNA binding peptides or proteins which may assist inbringing the endonuclease into juxtaposition with a target nucleic acidsuch that hybridization and cleavage of the target sequence may takeplace. Nucleotide sequences may be added to the 5' and 3' ends of thegroups X and Y to increase affinity for substrates. Such additionalnucleotide sequences may form triple helices with target sequences(Strobel, S. A., et al., (1991) Nature 350: 172-174 and referencestherein which are incorporated by reference) which may enableinteraction with intramolecularly folded substrate. Alternatively,modified bases (non-natural or modified bases as described in Principlesof Nucleic Acid Structure, Supra) bases within the additional nucleotidesequences may be used that will associate with either single stranded orduplex DNA generating base pair, triplet, or quadruplet, interactionswith nucleotides in the substrate. Suitable bases would include inosine,5'-methylcytosine, 5'-bromouracil and other such bases as are well knownin the art, as described, for example, in Principles of Nucleic AcidStructure, Supra.

In accordance with another aspect of this invention there is provided anendonuclease of the formula (I) as hereinbefore described wherein one ofsaid nucleotide sequences X or Y includes the target polynucleotidewhich is cleaved by the endonuclease. Such an embodiment may cause therelease of active RNA fragments on cleavage of a target RNA, whichfragments may themselves possess endonuclease activity.

In accordance with another aspect of this invention, there is provided apoly-endonuclease of the formula (IV):

    Y--M--Y-(X--M--Y).sub.n

where

X, Y and M are as previously defined, and

n is an integer of from 1 to 100.

Poly-endonucleases have the potential to act as anti-sense molecules(Helene, C. and J-J Toulme (1990) Biochemica, Biophysica, Acta 1049:99-125) as well as endonucleases. By "antisense" is meant the formationof a duplex or double stranded sequence as a result of base pairingbetween complementary bases of a target sequence and an antisenseoligonucleotide, which prevents translation of said sequence as a resultof duplex formation or the creation of a template for cleavage of theRNA by RNase H, a cellular ribonuclease which acts to cleave the RNAcomponent of hybridised RNA and DNA sequences. The possibility of actingas antisense may also arise when the groups X and Y of the endonucleaseof this invention contain a significant number of nucleotides, such as30 or more nucleotides. A duplex formed between such an endonuclease anda target sequence may not dissociate or readily melt under ambientconditions.

In accordance with yet another aspect of this invention, there isprovided a composition comprising an effective amount of an endonucleaseof the formula (I) as hereinbefore defined either alone or inassociation with one or more pharmaceutically, veterinarally oragriculturally acceptable carriers or excipients.

An effective amount or a therapeutically effective amount of anendonuclease of the formula (I) is an amount effective to cause cleavageof target RNA and/or inactivation thereof (such as by the provision ofan endonuclease of the formula (I) wherein the nucleotide sequences Xand Y comprise a significant number of nucleotides, such that theendonuclease essentially binds irreversibly to the target) so as toameliorate disease in a human, animal or plant subject. What constitutesan effective amount of an endonuclease will vary depending on the natureof the disease being treated, the mode of application of theendonuclease to the subject, health of the subject, weight of thesubject, and other like factors, as are well known in the pharmaceuticalart to be associated with pharmaceutical effectiveness (see ThePharmacological Basis of Therapeutics, 4th Edition, Lewis S. Goodman andAlfred Gilman, 1970, The Macmillan Company). What constitutes atherapeutically effective dose in any particular circumstance can bereadily determined according to standard procedures known in thepharmaceutical art (see The Pharmacological Basis of Therapeutics,Supra). For example, titration experiments may be employed where theeffect of an endonuclease in tissue culture is studied to determinetoxicity and effectiveness. Thereafter, trials may be conducted onanimals and thereafter human patients to determine toxicity,effectiveness, preferred mode of administration and the like. Suchinvestigations are routine in the pharmaceutical art as mentioned above,and thus a therapeutically effective amount of an endonuclease may bereadily determined without undue experimentation for medical orveterinary purposes.

A therapeutically effective amount of an endonuclease of the presentinvention would generally comprise from about 1 nM to about 1 mMconcentration in a dosage form, such as a cream for topical application,a sterile injectable composition, or other composition for parenteraladministration. In respect of topical formulations, it is generallypreferred that between about 50 μM to about 500 μM endonuclease beemployed. Endonucleases comprising nucleotide derivatives, whichderivatives may involve chemically modified groups, such asphosphorothioate or methyl phosphonate derivatives may be active innanomolar concentrations. Such concentrations may also be employed toavoid toxicity.

Therapeutic strategies involving treatment of disease employingendoribonucleases of this invention are generally the same as thoseinvolved with antisense approaches, such as described in the anti-sensebibliography of Chrisley, L. A. (1991) Antisense Research andDevelopment, 1: 65-113, which reference and all references therein areincorporated by reference. Particularly, concentrations of endonucleasesutilised, methods and modes of administration, and formulations involvedmay be the same as those employed for antisense applications.

By way of example only, therapeutic compositions of this invention maybe directed against Herpes Simplex virus types 1 and 2, psoriasis,cervical preneoplasia, papilloma disease, and bacterial and prokaryoticinfection. Such treatments may, for example, involve topical applicationof endonucleases to the site of disease. For example, in the treatmentof Herpes virus lesions, endonucleases may be formulated into a creamcontaining a concentration of 1 nM to 1 mM endonuclease. The cream maythen be applied to the site of infection over a 1 to 14 day period inorder to cause amelioration of symptoms of the infection. Prior to thefinal development of topical formulations for the treatment of Herpesvirus infection, effectiveness and toxicity of the endonucleases andformulations involving them may, for example, be tested on an animalmodel, such as scarified mouse ear, to which virus particles, such as2×10⁶ plaque forming units are added. A titre of infectious virusparticles in the ear after treatment can then be determined toinvestigate effectiveness of treatment, amount of nuclease required andlike considerations.

Similar investigations in animal models prior to human trialing may alsobe conducted, for example, in respect of the treatment of psoriasis,papilloma disease, cervical preneoplasia, and in diseases such as HIVinfection, bacterial or prokayrotic infection, viral infection andvarious neoplastic conditions, which neoplastic conditions involve adeleterious RNA species.

Pharmaceutically and veterinarally acceptable carriers and excipientsare well known in the art, and include carriers such as water, saline,dextrose and various sugar solutions, fatty acids, liposomes, oils, skinpenetrating agents, gel forming agents and the like, as described forexample in Remington's Pharmaceutical Sciences, 17th Edition, MackPublishing Co., Easton, Pa., Edited by Ostol et al., which isincorporated herein by reference.

Compositions for topical application are generally in the form ofcreams, where the endonucleases of this invention may be mixed withviscous components. In such embodiments, the endonucleases of thisinvention may be incorporated into liposomes or other barrier typepreparations to shield the endonucleases from nuclease attack or otherdegradative agents (such as endonucleases and adverse environmentalconditions such as UV light).

Compositions may be provided as unit dosages, such as capsules (forexample gelatin capsules), tablets, suppositories and the like.Injectible compositions may be in the form of sterile solutions ofendonuclease in saline, dextrose or other media. Compositions for oraladministration may be in the form of suspensions, solutions, syrups,capsules, tablets and the like. Endonucleases may also be provided inthe form of sustained release articles, impregnated bandages, patchesand the like. Pharmaceutical compositions which may be used in thisinvention are described, for example, in Remington's PharmaceuticalSciences, Supra.

The endonucleases of this invention may provided in a composition withone or more anti-viral, anti-fungal, anti-bacterial, anti-parasitic,anti-protazoan or anthelmentic agents, herbicides, pesticides or thelike, for example as described in the Merck Index (1989) 11th Edition,Merck & Co. Inc.

Agriculturally acceptable carriers and excipients are well known in theart and include water; surfactants; detergents; particularlybiodegradable detergents; talc; inorganic and/or organic nutrientsolutions; mineral earths and clays; calcium carbonate; gypsum; calciumsulfate; fertilisers such as ammonium sulfate, ammonium phosphate andurea; and natural products of vegetable origin such as, for example,grain, meals and flours, bark meals; and the like.

The endonucleases of this invention have extensive application, stemmingfrom the fact that virtually any RNA sequence may be cleaved by theendonucleases. The target nucleotide sequence GUC occurs, on a randombasis, approximately once every 64 bases. The general endonucleasecleavage site X'UY', wherein X' and Y' are any nucleotide, occurswherever the base uracil is present in an RNA, and thus any target RNAshould be cleavable, albeit at different efficiencies, using theendonucleases of this invention.

For in-vitro use, the endonucleases of this invention are generallyreacted with a target RNA which contains one or more suitable cleavagesites. Optionally, the target RNA may be purified or substantiallypurified. The nucleotide sequences X and Y of the endonucleases of thisinvention are selected so as to specifically hybridise or form adouble-stranded DNA duplex with a target RNA whereafter cleavage takesplace. Accordingly, target RNA may be specifically cleaved in-vitro inthe presence of other RNAs which themselves would not be cleaved by theendonucleases of this invention.

The endonucleases may be utilised in a manner similar to restrictionendonucleases, that is for the specific cleavage of RNA to facilitateRNA manipulation. All that is required for such manipulations is thatthe target RNA to be cleaved contains a uracil base and thus a suitablecleavage site.

Endonucleases of this invention may be utilised in diagnosticprocedures, such as the mapping or finger-printing of RNA. Specifically,the endonucleases of this invention would enable mapping of RNA and maybe used to detect mutations in RNA sequence. Such procedures may be usedin research and may also have forensic and other diagnosticapplications.

RNA cleavage products in-vitro may be readily detected, for example, byvisualisation on acrylamide or agarose gels where the amounts of RNAcleaved are sufficiently large for direct visualisation after separationand reaction with nucleotide visualisation agents, such as ethidiumbromide. Alternatively, where the target RNA cleaved is present in smallamounts, such as in a sample containing many RNAs, cleavage productsmay, for example, be detected by using radiolabelled probes of sequencecomplementary to the target sequence, or amplification techniques suchas PCR (Sambrook et al., Supra).

A target RNA for cleavage in-vitro may be derived from any source, andmay be of animal, viral, bacterial, plant, synthetic, or other origin.As RNA is common to all known living organisms, this invention may beutilised to cleave any RNA species having a suitable cleavage site asmentioned previously.

In-vitro cleavage of a target RNA is simply carried out by reacting thetarget RNA whether in purified, semi-purified or unpurified form, or asample containing the target RNA, with an effective amount of anendonuclease under reaction conditions facilitating RNA cleavage.Suitable reaction conditions include a reaction temperature of about 4°C. to about 60° C. (preferably about 20 to 55° C.), pH from about 7.0 toabout 9.0, and Mg²⁺ from about 1 mM to about 100 mM (preferably 1 to 20mM). The endonuclease may be present in an equimolar ratio to thesubstrate, or in excess thereof. As the endonucleases of this inventionmay act as enzymes, with each endonuclease cleaving multiple targetsequences, the endonuclease may be provided in less than an equimolarratio to target RNA.

According to an aspect of this invention, there is provided a method forthe cleavage of the target nucleotide sequence in-vitro which comprisesreacting said target nucleotide sequence or a sample containing saidtarget nucleotide sequence with an endonuclease as described hereinwherein nucleotide sequences X and Y of the nuclease are selected so asto be complementary to nucleotide sequences flanking a selected cleavagesite of the target RNA, such that on hybridisation of the endonucleaseto the target RNA, said target RNA is cleaved at the selected cleavagesite.

In circumstances where the nucleotide sequences X and Y comprise asignificant number of nucleotides, such as 30 or more nucleotides, theduplex formed on reaction of the endonuclease with its complementarytarget may not readily dissociate and hence such target RNAs may beinactivated not only by cleavage, but by blocking translation into adesired protein product, RNase H digestion, and/or prevention ofinteraction with other RNAs.

The endonucleases of this invention may be used for RNA cleavage in-vivoboth in prokaryotic and eukaryotic cells.

The endonucleases of this invention may be utilised to cleave any RNAwithin a cell which contains the cleavage site X'UY' as describedherein. Virtually all cellular RNAs would therefore be targetableutilising endonucleases of this invention.

Cleavage of target RNA within cells, such as bacterial cells, yeastcells, or animal cells, or the cleavage of a target RNA within the cellsof an organism, such as a plant or animal, may result in phenotypicmodifications or the treatment of disease or infection.

Phenotypic changes in plant cells or plants may include droughtresistance, salinity resistance, resistance to fungal, viral orbacterial infection; modifications of growth characteristics; sterility;fruit production; flowering; senescence and the like. It is evident thatonce one or more RNAs involved in determining phenotype are identified,such RNAs may be inactivated by cleavage utilising the endonucleases ofthis invention and thus the phenotype of the plant or plant cellaltered.

Phenotypic modifications within animals (including in some applicationsman) which may be effected by cleaving and thus inactivating target RNAsassociated with phenotype would include growth characteristics ofanimals, fertility, skin/cosmetic modifications, reproductivecharacteristics, disease resistance and the like. Myriad applicationsarise for phenotypic modifications in animals, and plants as previouslymentioned. Once one or more RNAs associated with a given phenotype areidentified and their sequence determined, endonucleases may be targetedagainst such RNAs for their inactivation with consequential phenotypicmodification.

Prokaryotic or eukaryotic cell cultures may be phenotypically modifiedby treatment with endonucleases of this invention. For example,bacterial cultures or yeast cultures involved in production of foodcomponents (such as cheese, bread and dairy products) and alcoholicbeverage production may be treated so as to modify enzyme content,flavour production, cell growth rate, culture conditions and the like.

The endonucleases of this invention may also be used to treat disease orinfection in humans, animals, plants, or prokaryotic or eukaryoticcells. The ability to treat disease or infection is based on the factthat the endonucleases of this invention are capable of cleaving any RNAwhich contains a suitable cleavage site, such as defined by the genericcleavage site X'UY', where X' and Y' represent any nucleotide(preferably wherein the cleavage site is GUC) as described previously.Most RNAs will contain one or more suitable cleavage sites.

The period of treatment would depend on the particular disease beingtreated and could be readily determined by a physician. Generallytreatment would continue until the disease being treated wasameliorated.

Examples of human and animal disease which may be treated with theendonucleases of this invention include Herpes Simplex Virus infection(such as targeting cleavage of early genes 4 and 5), psoriasis, cervicalpreneoplasia, papilloma disease, HIV infection (such as targeting theHIV-1 gag transcript and HIV-1 5'ltr splice site), bacterial andprokaryotic infection, viral infection and neoplastic conditionsassociated with the production of aberrant RNAs such as occurs inchronic myeloid leukemia. Diseases or infections which may be treated inplants with endonucleases of this invention include fungal infection,bacterial infections (such as Crown-Gall disease) and disease associatedwith plant viral infection.

Eukaryotic and prokaryotic cells in culture may, for example beprotected from infection or disease associated with mycoplasmainfection, phage infection, fungal infection and the like.

For the in-vivo applications of the endonucleases of this invention inhumans, animals, plants, and eukaryotic and prokaryotic cells, such asin phenotypic modification and the treatment of disease, it is necessaryto introduce the endonuclease into cells whereafter, cleavage of targetRNAs takes place.

Methods for the introduction of RNA and DNA sequences into cells, andthe expression of the same in prokaryotic and eukaryotic cells are wellknown in the art for example as discussed in Cotten, M. (1990) Tibtech8: 174-178; and Friedman, T. (1989) Science 244: 1275-1280 (both ofwhich references are incorporated herein by reference. The same widelyknown methods may be utilised in the present invention.

The endonucleases of this invention may be incorporated into cells bydirect cellular uptake, where the endonucleases of this invention wouldcross the cell membrane or cell wall from the extracellular environment.Agents may be employed to enhance cellular uptake, such as liposomes orlipophilic vehicle, cell permeability agents, such as dimethylsulfoxide,and the like.

Endonucleases of this invention may be incorporated and expressed incells as a part of a DNA or RNA transfer vector, or a combinationthereof, for the maintenance, replication and transcription of theendonuclease sequences of this invention.

Transfer vectors expressing endoribonucleases of this invention may becapable of replication in a host cell for stable expression ofendonuclease sequences. Alternatively, transfer vectors encodingendonuclease sequences of this invention may be incapable of replicationin host cells, and thus may result in transient expression ofendonuclease sequences. Methods for the production of DNA and RNAtransfer vectors, such as plasmids and viral constructs are well knownin the art and are described for example by Sambrook et al. (Supra).

Transfer vectors would generally comprise the nucleotide sequenceencoding the endonuclease of this invention, operably linked to apromoter and other regulatory sequences required for expression andoptionally replication in prokaryotic and/or eukaryotic cells. Suitablepromoters and regulatory sequences for transfer vector maintenance andexpression in plant, animal, bacterial, and other cell types are wellknown in the art and are described for example in Hogan, B. et al.,(1986) Manipulating the Mouse Embryo, A Laboratory Manual, Cold SpringHarbor; and Science (1989) 244: 1275-137, which are incoporated hereinby reference.

Transfer vectors or nucleic acid sequences encoding or comprising theendonucleases of this invention may be incorporated into host cells,such as plant or animal cells, by methods well known in the art (forexample, as described by Cotten and Friedman (Supra), such asmicroinjection, electroporation, receptor-mediated endocytosis,transformation of competent cells such as protoplasts or bacterial cellstreated with metal ions such as calcium chloride, cationic or otherliposomes, viral or pseudovirus vectors, DEAE-Dextran, or by usingprojectiles to penetrate cell walls and thereby deliver the desirednucleic acid sequence.

In accordance with a still further aspect of this invention, there isprovided a transfer vector which encodes a nucleotide sequences encodingan endonuclease as described herein.

Nucleotide sequences encoding the endonucleases of this invention may beintegrated into the genome of a eukaryotic or prokaryotic host cell forsubsequent expression (for example as described by Sambrook et al.,Supra). Genomic integration may be facilitated by transfer vectors whichintegrate into the host genome. Such vectors may include nucleotidesequences, for example of viral or regulatory origin, which facilitategenomic integration. Methods for the insertion of nucleotide sequencesinto a host genome are described for example in Sambrook et al. andHogan et al., Supra.

Genomically integrated nucleic acid sequences encoding the endonucleasesof this invention generally comprise a promoter operably linked to thenucleotide sequence encoding the endonuclease of this invention, andcapable of expressing said endonuclease in a eukaryotic (such as animalor plant cells) or prokaryotic (such as bacteria) host cells.

Endonucleases of this invention may be involved in gene therapytechniques, where, for example, cells from a human suffering from adisease, such as HIV are removed from a patient, treated with theendonuclease to inactivate the infectious agent, and then returned tothe patient to repopulate a target site with resistant cells. In thecase of HIV, nucleotide sequences encoding endonucleases of thisinvention capable of inactivating the HIV virus may be integrated intothe genome of lymphocytes or be present in the cells a transfer vectorcapable of expressing endonucleases of this invention. Such cells wouldbe resistant to HIV infection and the progeny thereof would also confersuch resistance.

In accordance with an aspect of this invention, there is provided amethod for the cleavage of a target nucleic acid sequence either in-vivoor in-vitro which comprises reacting a target nucleotide sequence withan effective amount of an endonuclease as described herein, saidendonuclease being capable of effecting specific cleavage of said targetat a site selected such as to cleave and inactivate the target nucleicacid sequence.

In accordance with another aspect of this invention, there is provided amethod for the treatment of disease or infection in a human, animal,plant, or prokaryotic or eukaryotic cell, which is associated with thepresence of a deleterious RNA, which method comprises treating saidhuman, animal, plant, prokaryotic or eukaryotic cell with an effectiveamount of an endonuclease as described herein, either alone or inassociation with a pharmaceutically, veterinarally, or agriculturallyacceptable carrier or excipient, which endonuclease is capable ofcleaving and thus inactivating said deleterious RNA.

Recombinant DNA manipulations referred to above are well known in theart, and are described for example by Sambrook et al., Supra.

In another aspect of this invention, there is provided an animal orplant which comprises one or more cells which have been modified by, orcontain, encode and/or express an endonuclease as herein defined.

The endonucleases of this invention may be produced by nucleotidesynthetic techniques which are well known in the art, and described forexample by Carruthers et al. (Methods in Enzymology (1987) 154:287-313), Foehler et al. (Nucleic Acids Resarch (1986) 14: 5399-407) andSprat et al. (Oligonucleotide Synthesis--A Practical Approach, IRLPress, Oxford (1984) M. J. Gait--Editor, pp. 83-115), all of which areincorporated herein by reference. Generally, such synthetic proceduresinvolve the sequential coupling of activated and protected nucleotidebases to give a protected nucleotide chain, whereafter protecting groupsmay be removed by suitable treatment. Alternatively, the endonucleasesin accordance with this invention may be produced by transcription ofnucleotide sequences encoding said endonucleases in host-cells or incell free systems utilizing enzymes such as T3,SP6 or T7 RNA-polymerase(Sambrook et al., Supra).

The catalytic region M of the endonucleases of this invention is of areduced size compared with what may have been considered necessary fromknowledge in the prior art. The absence of a conventional base-pairedstem structure provided by an embodiment of the endonucleases of thisinvention may reduce steric interference of the endonuclease with itssubstrate or other nucleic acid structures and may also reduce thelikelihood of non-favourable base pairing interactions particularly inthe in-vivo context when the endonuclease may be in association with alarge number of nucleic acids, in addition to the specific target whichit is engineered to cleave.

The inclusion of deoxyribonucleotides in the endonuclease structure incertain embodiments of this invention may provide protection againstribonuclease degradation. Also, endonucleases comprised of RNA/DNA maynot provide a substrate for unwinding/modifying enzymes whichcompromises some anti-sense applications. The reduced size of variousembodiments of the endonuclease of this invention when compared withother endonucleases known in the art may serve to improve the economicsof synthesis of the endonuclease and may also serve to improve the easeand efficiency of the introduction of the endonuclease into host cells.

Poly-endonucleases as described herein may be designed to have cleavagesequences within the flanking regions X and Y which link catalyticdomains. Such poly-endonucleases may autocatalytically liberate multipleindividual endonucleases in cells, thus increasing the localconcentration of endonucleases.

The oligonucleotide backbone (that is, phosphodiester linkages) ofcompounds of the formula (I) may be modified in a variety of ways, forexample, in the same manner as for DNA antisense oligonucleotides.Methylphosphonate, phosphorothioate and phosphoramidate linkages may beused to replace conventional phosphodiester linkages. Additionally,ribonucleotides may be substituted with modified nucleotides and/orbases, for example, 2'methoxyribonucleotides or [α]-anomers as describedherein. These modifications may confer nuclease resistance and improvebiological half-life and/or cellular uptake of endoribonucleases.Phosphorothioate linkages confer RNAse H sensitivity to the RNAcomponent of RNA/DNA duplexes. Methylphosphonate linkages confer RNase Hresistance to this same component.

Various embodiments to the present invention will now be described, byway of non-limiting example only, in the following examples.

EXAMPLE 1 Endonuclease Synthesis

All endonucleotides were synthesized on either or both an AppliedBiosystems 380 or 391 synthesizer using 2-cyanoethylphosphoramiditechemistry. DNA monomers and RNA monomers, protected at the 2' positionwith a t-butyldimethylsilyl group, were obtained from commercialsuppliers. All oligonucleotides, with 5'-trityl groups removed, wereworked up as follows: the oligonucleotide was cleaved from the column in3:1 NH₄ OH/ethanol, and heated overnight at 55° C. The solutions wereevaporated to near dryness, taken up in H₂ O/ethanol 3:1 and dried, andrepeated. The amount of material was estimated at this stage bymeasuring the UV absorbance. The 2' group was deprotected by treatmentovernight with 1 M tetrabutylammonium fluoride in THF (10 μL per OD₂₆₀).The tetrabutylammonium ions were removed by passage twice through aDowex 50X8-200 (trade mark) cation-exchange column in the Na⁺ form, thevolume of the eluate was reduced with 2-butanol, and the oligonucleotideprecipitated with sodium acetate and ethanol. The oligonucleotide wasthen purified by electrophoresis on a 10-20% (depending on length)acrylamide gel containing 7 M urea. The band of interest was visualisedby UV shadowing or ethidium bromide staining, excised and soaked inwater. The oligonucleotide solution was removed from the gel slices,concentrated with 2-butanol, washed with phenol/chloroform and ether.The oligonucleotide was then precipitated with sodium acetate andethanol, washed with cold 80% ethanol, redissolved in 10 mM Tris-Cl, pH8.0, 2 mM EDTA, quantified by UV spectroscopy, and frozen. Purity of theoligonucleotides was determined by labelling the 5' end with ³² Pphosphate and running out in a denaturing gel. Oligonucleotides werephosphorylated using standard conditions, except that several units ofpancreatic ribonuclease inhibitor were added to the reaction mixture.Concentration of labelled material was determined by pooling all wastefrom the phosphorylation procedure, drying down, and running on a gelalongside a known fraction of the labelled oligonucleotide; and bandswere excised and counted, and from this the amount of material lost inthe phosphorylation procedure was known, and the concentration of thelabelled oligonucleotide determined.

Reaction Conditions

Typically reactions were conducted in 50 mM Tris.HCl, pH 8.0, 10 mMMgCl₂ at 37° C.; substrate concentration was 100 nM, and endonucleaseconcentration was 100 or 600 nM in all reactions. The reactions wereconducted in a 30 μL volume over a range of 30 minutes to 4 hours. Themolar ratio of endonuclease to substrate was 1:1 or 6:1. Theendonuclease and substrate (³² P labelled) were heated separately inreaction buffer to 70° C. for three minutes, then snap-cooled beforemixing and subsequent incubation. Departures from the standard reactionconditions were taken as needed in experiments aimed at determining thetemperature/activity profile, magnesium dependency, and pH dependencyand turnover of the endonuclease mediated cleavage reaction. Sampleswere then analysed by electrophoresis in 15% acrylamide gels containing7 M urea as a denaturant. The substrate and product of cleavage werevisualised by autoradiography, and gel slices corresponding to theirpositions were excised and quantified by Cerenkov counting.

EXAMPLE 2 Growth Hormone RNA Targeted Endonucleases

Base sequence, schematic representation and names of variousendonucleases are set out below. Conserved ribonucleotides are depictedby bold type and thick lines. Other ribonucleotides are depicted byupper case letters and thin lines. Deoxyribonucleotides are depicted bylower case letters and wavy lines. The nomenclature for theendonucleases is as follows: R indicates a ribozyme containing helix II,M denotes an endonuclease not containing helix II, and MS denotes aendonuclease and substrate in the same molecule; the numbers andnucleotide designations indicates the bases in the connector, and theRNA or DNA refers to the nucleotides in the arms that form helices I andIII with the substrate. Double helices I, II and III are as described byForster and Symons (Cell (1987), 49: 211-220).

A further series of growth hormone RNA targeted endonucleases weresynthesised based on the M4t,DNA construct and having different numbersand types of nucleotides in the connector. These are as follows:

M2t,DNA

M3t,DNA

M5t,DNA

Mttct,DNA

MttPDt,DNA

PD refers to 1,3-propanediol which was used in place of a nucleoside.

An endonuclease R4U,DNA was also synthesized. This endonuclease is thesame as R4U,RNA depicted above, except that the RNA flanking sequencesare replaced with DNA.

SUBSTRATES

Rat Growth Hormone 21 Mer's:

All of the above endonucleases were reacted with a ribonucleotidesequence corresponding to a portion of the rat growth hormone gene andhaving the sequence (SEQ ID NO:8): ##STR3##

A second substrate (GHS2) corresponding to GHS1 but where allnucleotides except C* were deoxyribonucleotides was also synthesized.Endonucleases M4U,RNA and M4U,DNA were reacted with this substrate.

Kruppel RNA:

The endonuclease M4t,DNA, Kr1079 (comprising 34 nucleotides and havingflanking sequences of DNA designed to hybridise to the Kruppel targetRNA) was tested against a short synthetic RNA substrate of 21nucleotides and a RNA substrate of approximately 1.9 Kb, both containingthe same cleavage site.

The Kr RNA transcript was prepared by inserting cDNA encoding the Krtranscript into a plasmid containing the T7RNA polymerase promoter. TheKr transcript was then transcribed with T7-polymerase.

The synthetic 21 mer was chemically synthesised and contained the samecleavage site as the longer RNA transcript. The 21 mer comprised thefollowing sequence: ##STR4## where C* is a ribonucleotide.

In-vivo testing of activity of an anti-Kruppel endonuclease could beaccomplished by microinjection of Drosophila embryos prior to the stageof syncytial blastoderm, in order to inactivate the 2.3 Kb RNA Kruppeltranscript. Embryos (cuticlised embryos) can be assayed for abberantsegmental pattern one to two days after egg laying.

Platelet Derived Growth Factor (PDGF):

The endonuclease Mttct,DNA, PDGF (a 30 mer, having flanking DNAsequences designed to hybridise to the PDGF target RNA) was reacted witha 666 base RNA transcript corresponding to exons 2 and 6 of the PDGF Agene from humans. A 666 base pair RNA transcript was transcribedin-vitro from a PDGF gene fragment using T3 polymerase.

RESULTS

A. All growth hormone RNA targetted endonucleases cleaved GHS1 withvarying activities. In keeping with the endoribonuclease of Haseloff andGerlach (Supra) and those of other investigators, no endonucleasecleaved 100% of product over the reaction period. Endonucleases M4T,DNA;M4U,RNA; M4U,DNA; M4A,DNA; M3T,DNA; M5T,DNA; and MTTCT,DNA; all cleavedbetween about 50-60% of substrate over the reaction period, this beingabout the same as the control R4U,RNA. Endonucleases containing lessthan four nucleotides in the connector (corresponding to the group P ofthe formula (II)) were somewhat less active in the test assay.

Experiments were conducted to examine the enzymatic turn over,temperature activity profile, magnesium dependency and pH dependence ofrepresentative minizyme M4T,DNA and a reference control, R4U,RNA,corresponding to the endoribonuclease of Haseloff and Gerlach (Supra).These experiments showed that both endonucleases behaved as enzymesexhibiting turn over, achieving cleavage of target substrate at pH 7.5,37° C. and greater than or equal to 1 mM Mg2+.

It is clear from these results that functional endonucleases capable ofcleaving target substrates may contain hybridising arms comprised of DNAor RNA, combinations thereof (corresponding to the groups X and Y offormula (I)); a connector comprised of a non-base paired RNA or DNAsequence, or a partly base paired RNA sequence and that the number andnature of nucleotides in the connecting sequence is not of importance.

The endonuclease MttPDt,DNA was active and shows that nucleotides may bereplaced with chemical linking groups without loss of endonucleaseactivity. On the basis of this experiment, it is apparent thatnucleotides in the connector sequence (corresponding to the group P ofthe formula (II)) may be replaced with linker sequences.

B. The substrate GHS2 was cleaved by M4U,RNA but not M4U,DNA. It ispresently unclear why the M4U,DNA endonuclease was not active againstthis substrate although this was obviously suggestive that the structureof a DNA-DNA duplex is sufficiently different from that of an RNA-RNA oran RNA-DNA duplex of the same sequence so as to possibly alter thestructure of the active site and render the endonuclease inactive.

As the endonuclease M4U,RNA cuts a substrate made entirely of DNA,except for the central ribonucleotide, it is apparent that the only 2'hydroxyl on the substrate required for the cleavage reaction is on thenucleotide to be cleaved.

By altering the nucleotide composition of the flanking sequences of theendonuclease (corresponding to the groups X and Y of the formula (II)),any nucleotide substrate containing a suitable cleavage site may becleaved. Optimisation of the flanking nucleotides as hereinbeforedefined may be required to facilitate cleavage, as illustrated by thefact that M4U,RNA cleaves a substrate made entirely of DNA except forthe central ribonucleotide having the base C, whereas the endonucleaseM4U,DNA does not cleave this substrate.

Cleavage of the 1.9Kb base Kruppel RNA and synthetic 21 mer RNA targethybrid was observed with the M4t,DNA,Kr1079 endonuclease.

Cleavage of the 1.9 Kb Kruppel RNA transcript indicates that large RNAscan be selectively cleaved at a desired cleavage site.

C. Similarly, an individual endonuclease effected efficient and specificcleavage of the long PDGF RNA transcript.

A synthetic RNA linked in cis to its target sequence, MS4U,RNA, was alsoactive in effective specific cleavage at the predicted target sequence,at nanomolar concentrations with a rate independent of concentration.This provides evidence that this endonuclease does not form polymers orhigh molecular weight structures to effect cleavage, but rather that anindividual discrete nuclease is capable of effecting catalytic cleavageof a target nucleotide sequence.

It is clear from the above examples that the endonucleases of thisinvention may be selectively used for the cleavage of any target RNAwhich contains an appropriate cleavage site (such as GUC) and whosenucleotide sequence is known. Accordingly, the endonucleases of thisinvention have wide application in the selective inactivation of targetRNAs in-vivo and in-vitro and as such may be used for example for thetreatment of disease in humans and animals, phenotypic alteration inanimals and plants and other myriad applications.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 9                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: RNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ACCUGCGGGU CAUGAAGUGU C           - #                  - #                      - #21                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other nucleic acid                                         (A) DESCRIPTION: (Mixed - #DNA/RNA oligomer                                        see speci - #fication and figures for details)                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - GACACUUCAU CUGAUGAGUC CUUUUGGACG AAACCCGCAG GU    - #                      - #  42                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other nucleic acid                                         (A) DESCRIPTION: (Mixed - #DNA/RNA oligomer                                        see speci - #fication and figures for details)                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GACACUUCAU CUGAUGAUUU UGAAACCCGC AGGT       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other nucleic acid                                         (A) DESCRIPTION: (Mixed - #DNA/RNA oligomer                                        see speci - #fication and figures for details)                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - GACACTTCAT CUGAUGAUUU UGAAACCCGC AGGT       - #                  -     #        34                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other nucleic acid                                         (A) DESCRIPTION: (Mixed - #DNA/RNA oligomer                                        see speci - #fication and figures for details)                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GACACTTCAT CUGAUGATTT TGAAACCCGC AGGT       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: RNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - GACACTTCAT CUGAUGAAAA AGAAACCCGC AGGT       - #                  -     #        34                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: RNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - GCGGGUCAUG AAGUUCGCUU CAUCUGAUGA UUUUGAAACC CG    - #                      - #  42                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other nucleic acid                                         (A) DESCRIPTION: (Mixed - #DNA/RNA oligomer                                        see speci - #fication and figures for details)                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - ACCUGCGGGU CAUGAAGUGU C           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: RNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - AUUUGCGAGU CCACACUGGA G           - #                  - #                      - #21                                                                 __________________________________________________________________________

We claim:
 1. A compound comprising the structure: ##STR5## wherein eachX represents a ribonucleotide or a deoxyribonucleotide which is the sameor different and may be modified or substituted in its sugar, phosphateor base;wherein each of A, C, U, and G represents a ribonucleotide;wherein N represents any ribonucleotide; wherein each one of (X)_(n) and(X)_(n), represents an oligonucleotide comprising at least onedeoxyribonucleotide, in which n and n' are integers which define thenumber of nucleotides in the oligonucleotide, such oligonucleotidehaving a predetermined sequence sufficiently complementary to apredefined RNA target sequence to allow hybridization to the RNA targetsequence; wherein each solid line represents a covalent bond between thenucleotides located on either side thereof; wherein m represents aninteger form 2 to 20; and wherein none of the nucleotides (X)_(m) areWatson-Crick base paired to any other nucleotide within (X)_(m).
 2. Thecompound of claim 1, wherein each X represents a deoxyribonucleotide. 3.The compound of claim 1, wherein m is
 4. 4. The compound of claim 1,wherein N represents U, a ribonucleotide having a uracil base.